Antigens are processed by acidic proteases prior to presentation in the context of class II histocompatibility proteins. The formation of a trimolecular complex of processed antigen, class II proteins, and T-cell receptor is essential for immune recognition and responses. This study will investigate the role of acidic endosomal proteases in antigen processing and invariant chain release from HLA class II proteins. The coordinate regulation of these proteolytic events is critical to antigen presentation, as invariant chain release must occur prior to peptide antigens binding to class II proteins. In humans, the precise enzymes which function in antigen and invariant chain processing have not been identified nor have the factors which regulate protease activity been defined. Immune responsiveness may be linked to protease expression, as differences in antigen processing have been detected among individuals. Proteolytic processing is a prerequisite for the presentation and recognition of both foreign and self antigens. The generation of self peptides which bind specific class II alleles, may be a key event initiating autoimmune reactivity. Thus, protease expression may represent a previously unrecognized genetic factor in susceptibility to autoimmune disorders. The long-term goals of this research program are to define the critical intracellular events which regulate antigen presentation and determine how protease expression controls responses to foreign and self antigens in humans. For these purposes, antigen processing and presentation will be studied in human B-cell lines which display high levels of class II proteins. In B-cells both invariant chain release from class II proteins and antigen processing may occur in endosomes. Specifically, this proposal will: 1) Determine if antigenic peptides are generated by endosomal proteases and how changes in endosomal protease activity modulate antigen presentation; 2) Evaluate if polymorphic class II antigens found in endosomes influence processing and the formation of antigenic determinants; 3) Investigate the requirement for aspartyl proteases, such as cathepsin D in the processing of antigens and invariant chain through the generation of mutant cell lines. A combination of cell biological as well as molecular and cellular immunological approaches will be employed for each of these specific aims.